Hemolytic anemia 3

HEMOLYTIC ANEMIAS
Prof. Dr. Khalid Amin
Head of Medical Unit-IV
Faisalabad Medical University
Allied/DHQ Hospitals,Faisalabad

Hemolytic Anemias
Definition
 A group of disorders leading to anemia
caused by a reduction in red cell life span.
 Anemia is the result of premature destruction
of red cells exceeding the erythropoietic
capacity of the bone marrow.
 Bone marrow has the capacity to increase
erythropoiesis 6 - 8 times than normal.

Hemolytic Anemias
Classification
Hemolytic anemias may be classified as:
 I- Hereditary or Acquired
or
 II- Intracorpuscular or Extracorpuscular

Hemolytic Anemias
Classification
Hemolysis may occur in two compartments:
 I- Intravascular
or
 II- Extravascular ( eg; spleen )

1- Abnormalities of RBC interior
a. Enzyme defects
b. Hemoglobinopathies & Thalassemia Major
2- RBC membrane abnormalities
a. Hereditary spherocytosis, elliptocytosis etc
b. Paroxysmal nocturnal hemoglobinuria
c. Spur cell anemia
Hemolytic Anemias
Classification

3- Extrinsic factors:
a. Hypersplenism
b. Antibody : immune hemolysis
c. Traumatic & Microangiopathic hemolysis
d. Infections , toxins , etc
Hemolytic Anemias
Classification

PATHOPYSIOLOGY
Extravascular Intravascular
RES
Haem
Globin
Plasma
iron pool
Plasma
protein pool
Protoporphyrin
Expired CO Unconjugated
bilirubin
Liver
Conjugated
bilirubin
GI tract
Urobilinogen
Faeces
Urine
Free plasma Hb
Hb- Hpt complex
Liver
Hpt and
Hpx
Haemopexin-
methem
Excess Hb
methaemalbumi
n
Kidney
Hb Haemosiderin
Haem+globin
Hb
methem
metHb

Clinical Manifestations
in Summary
 Onset may be acute or insidious
 Symptoms and signs of Anemia
 Jaundice
– Acholuric
– Without pruritus
 Symptoms and signs spesific to the type of
hemolytic anemia
 Symptoms related to the underlying disease

in Summary
 Splenomegaly
– Most congenital hem. anemias except sickle
cell
– Some of the acquired hem. anemias
 Cholelithiasis (gall stones) symptoms
 Leg ulcers (sickle cell, spherocytosis)

in Summary
 Skeletal abnormalities (thalassemia)
 Crises (chronic hemolytic disease)
– Aplastic crises (HPV-B19)
– Hemolytic
– Megaloblastic
 Changes in urine color

Laboratory Findings
Increased RBC Destruction
 Decreased RBC life span
 Increased haem(heme) catabolism
– Increased serum unconjugated bilirubin
– Increased endogenous CO production
– Increased urobilinogen excretion
 Increased serum LDH
 Absence or decrease of serum haptoglobin

Laboratory Findings
Increased RBC Destruction
 > 1 g /dl /week fall in blood Hb level
 Reduced glycosylated Hb (HbA1C)
 Signs of intravascular hemolysis
– Hemoglobinemia
– Hemoglobinuria
– Hemosiderinuria
– Methemalbuminemia
– Reduced serum hemopexin level

Laboratory Findings
Increased Bone Marrow Activity And RBC Production
 Blood
– Reticulocytosis
– Macrocytosis
– Polychromatophilia
– Erythroblastosis
– Leukocytosis and thrombocytosis
 Bone marrow
– Erythroid hyperplasia

Laboratory Findings
Increased Bone Marrow Activity And RBC Production
 Ferrokinetic
– Increased plasma iron turnover
– Increased RBC iron turnover
 Biochemical
– Increased RBC creatine
– Increased activity of RBC enzymes eg: hexokinase,
etc

Laboratory Evaluation of Hemolysis
Extravascular
Hematologic
 Blood film Polychromatophilia
 Reticulocyte Increased
 Bone marrow Erythroid hyperplasia
Plasma or serum
 Bilirubin unconjugated
 Haptoglobin , absent
 Plasma free Hb N -
 LDH
Urine
 Bilirubin 0
 Hemosiderin 0
 Hemoglobin 0
 Urobilinogen
Intravascular
Polychromatophilia
Increased
Erythroid hyperplasia
unconjugated
absent
0
+
+

Laboratory Tests Useful İn Differential
Diagnosis
 Examination of Peripheral Blood
 Special Lab. Examinations

Morphologic Abnormalities in
Hemolytic Anemias
 Polychromasia :
– Reticulocytes
 Spherocyte :
– Her. Spherocytosis, immune hem. Anemia,burns,
chemical injury to RBC
 Acanthocytes :
– Spur cell anemia with liver disease
– abetalipoproteinemia
– Pyruvate kinase deficiency
– uremia

Hemolytic Anemias
 Sickle cell:
– Sickle cell anemia
 Target cels:
– Thalassemia
– liver disease, Splenectomy
 Schistocytes:
– Microangiopathic hem anemia, uremia, DIC,
– malignant hypertesion, eclampsia,

Hemolytic Anemias
 Agglutination
– Cold agglutinin disease
 Heinz bodies:
– Unstable Hb, G6PD deficiency and oxidant stress
 Howell-Jolly bodies
– Splenectomy, Hyposplenism
– Megaloblastic anemia, Hemolytic anemia

Hemolytic Anemias
 Basophylic stippling
– Lead poisoning
– Thalassemia,
– Unstable hemoglobines
– MDS
– Megaloblastic anemia

Special Lab. Examinations
 Coombs Antiglobulin Test
– immune hemolysis
 Osmotic fragility test
– spherocytosis
 Autohemolysis
– G6PD,PK, spherocytosis
 Membrane protein analysis
– membrane defects
 Red cell sickling
– sickle cell anemia

Special Lab. Examinations
 Hemoglobin electrophoresis and HbA2, Hb F ,
HHb,etc
– Hemoglobinopathies and thalassemias
 Red cell enzyme assays
– RBC enzyme defects
 HAM and sucrose lysis tests and GPI-linked protein
analysis by flow cytometry
– PNH
 Oxygen dissociation curve
– High oxygen affinity Hb

Red Cell Membrane Disorders
 Usually detected by morphologic abnormalities of
RBC on blood film.
 Three types of inherited RBC membrane
abnormalities:
– Hereditary Spherocytosis.
– Hereditary Elliptocytosis (Including Hereditary
Pyropoikilocytosis).
– Hereditary Stomatocytosis.

Red Cell Membrane Disorders
 Red cell membrane has a lipid bilayer to which a
‘skeleton’ of filamentous proteins is attached via
special linkage proteins.

HEREDITARY SPHEROCYTOSIS
“ Defective or absent spectrin molecule”
 Leads to loss of RBC membrane, leading to
spherocytosis
 Decreased deformability of cell
 Increased osmotic fragility
 Extravascular hemolysis in spleen
 Usually has an autosomal dominant
inheritance

 Clinical Manifestations
– Anemia.
– Splenomegaly.
– Jaundice.
– Jaundice may be intermittent and tends to be less
pronounced in early childhood.
– Due to increased bile pigment production, pigmented
gallstones are common, even in childhood.

 Diagnosis
 Osmotic Fragility Test
0
20
40
60
80
100
0.3 0.4 0.5 0.6
NaCl (% of normal saline)
%
Hemolysis
Normal HS

Treatment
– Splenectomy reliably corrects the anemia.
– Splenectomy in children should be
postponed until the age of 4-years.
– Polyvalent pneumococcal vaccine
should be administered at least 2-weeks
before splenectomy
– Folic acid (5-mg/d) should be given
prophylactically.

Hereditary Elliptocytosis
 Heterogeneous disorders in elliptocytic red cells.
 Functional abnormality of anchor proteins in red
cell membrane (alpha spectrin or protein 4.1).
 Autosomal dominant or recessive.

Hereditary Elliptocytosis
 Clinical presentation depends upon degree of
membrane defect.
 Asymptomatic diagnosed on blood film.
 Chronic compensated hemolytic state.
 Treatment
– Same as hereditary spherocytosis only in
chronic hemolytic anemia.

Paroxysmal Nocturnal
Hemoglobinuria (PNH)

Paroxysmal Nocturnal
Hemoglobinuria (PNH)
 Acquired deficit of GPI-Associated proteins
 Ongoing Intra- & Extravascular hemolysis;
classically at night
 Testing
– Acid hemolysis (Ham test)
– Sucrose hemolysis
– CD-59 negative (Product of PIG-A gene)

HEMOLYTIC ANEMIA
Membrane Abnormalities - Enzymopathies
 Deficiencies in Hexose Monophosphate
Shunt (HMP Shunt)
– Glucose 6-Phosphate Dehydrogenase
Deficiency
 Deficiencies in the EM Pathway
– Pyruvate Kinase Deficiency

GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY
(G6PD DEFICIENCY)

(G6PD DEFICIENCY)
 Glucose-6-phosphate dehydrogenase deficiency is
an X-linked Recessive Hereditary Disease
charachterised by abnormally low levels of
glucose-6-phosphate dehydrogenase.
 G6PDH is an metabolic enzyme involved in the
pentose pathway, important pathway in red blood
cell metabolism.
 G6PDH is the most common human enzyme
defect.

(G6PD DEFICIENCY)
PRECIPITATING FACTORS
 Antimalarials:
Primaquine,
Quinine,
Chloroquine
 Antibiotics:
Nitrofuantoin,
Furazolidine,
Cotrimoxazole,
Nalidixicacid,
Chloramphenicol,

(G6PD DEFICIENCY)
PRECIPITATING FACTORS
 Others :
– Vitamin K – large doses
– Naphthalene
– Benzene
– Methylene blue
– Probenecid
– Acetyl salicylic acid (aspirin)
– Fava beans
– Diabetic ketoacidosis

(G6PD DEFICIENCY)
Exposure to drug/precipitant
24-48 hr
 Severe progressive anemia, cardiac failure and
jaundice
 Favism: hemolysis after ingestion of fava beans

(G6PD DEFICIENCY)
Clinical Features:
 Paleness
 Extreme Tiredness
 Rapid Heartbeat
 Shortness of Breath
 Jaundice, or yellowing of the skin and eyes
 Enlarged Spleen
 Dark, Tea-Colored Urine

(G6PD DEFICIENCY)
INVESTIGATIONS
During hemolysis :
 Hb decreased
 Elevated reticuloycytes (5-15 %)
 Perpheral Smear:
Normocytic normochromic anemia
Polychromasia, fragmented cells
Heinz bodies
 Hemoglobinuria
 Indirect hyperbilirubinemia

(G6PD DEFICIENCY)
MANAGEMENT
 Prevention of haemolysis by prompt treatment
of infection.
 Avoidance of oxidant drugs and toxin
 Avoid the oxidants
 Blood transfusion
 Sodium bicarbonate to alkalinise urine in
severe Hemoglobinuria

AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA)

General Principles
 All require Antigen-antibody Reactions
 Types of reactions dependent on:
– Class of Antibody
– Number & Spacing of antigenic sites on cell
– Availability of complement
– Environmental Temperature
– Functional status of reticuloendothelial system

General Principles
 Manifestations
– Intravascular hemolysis
– Extravascular hemolysis

Types
 Warm Autoimmune Hemolytic Anemia (AIHA)
 Cold Autoimmune Hemolytic Anemia (AIHA)

WARM AUTOIMMUNE HEMOLYTIC ANEMIA
(AIHA)
 Due to development of an IgG antibody
 Antibody active at warm temperature of 37°C
 May be either primary or secondary in etiology
 Primary-
– idiopathic in nature
 Secondary-due to an underlying disease
– Lymphoproliferative disorders
– Autoimmune diseases
– Infection
– Immunodeficiency disorder
– Tumor

WARM AUTOIMMUNE HEMOLYTIC
ANEMIA (AIHA)
Epidemiology
 Incidence of 1-3 cases per 100,000 per year
 Favors females (female: male 2:1)
 Age at time of occurrence dependent on cause
– Primary more common in women during
the 4-5th decades of life
– Secondary occurs in association with
underlying disease

WARM AUTOIMMUNE HEMOLYTIC ANEMIA
(AIHA)
Mechanism of destruction
 Primarily due to Extravascular Hemolysis
 Antibodies bind to surface of RBC membrane
 Fc portion of antibody binds to macro-phages
– Interaction → spherocytes
 Spherocytes become trapped in spleen and are
destroyed

COLD AUTOIMMUNE HEMOLYTIC
ANEMIA (AIHA)
 Due to development of an IgM antibody
 Antibody active at cold temperature (4°C) and not
usually physiologically significant
 Either primary or secondary in etiology
– Primary-idiopathic in nature
– Secondary-due to an underlying disease
 Lymphoproliferative disorders
 Infection
 Autoimmune diseases
 Immunodeficiency disorder
 Tumor

ANEMIA (AIHA)
Epidemiology
 Accounts for 16-32% of cases of AIHA
 Affects older adults approximately 70 years of age
 Slight female predilection

ANEMIA (AIHA)
Mechanism of destruction
 Intravascular hemolysis
– IgM antibodies activate the compliment
system resulting in cytolysis
 Extravascular hemolysis
– C3b and iC3b rather than the Fc portion of
IgM are recognized
– Hemolysis occurs in the liver via action of
Kupffer cells

AUTOIMMUNE HEMOLYTIC ANEMIA
(AIHA)
Diagnosis
 Clinical signs and symptoms
– Fatigue
– Pallor
– Jaundice
– Shortness of breath
– Heart failure
– Raynaud’s phenomenon, vascular
occlusions/necrosis in cold agglutinin disease

(AIHA)
Diagnosis
 Lab data
– CBC with peripheral smear
– LDH
– Haptoglobin
– Bilirubin

(AIHA)
Diagnosis
 Disease severity depends on degree of hemolysis
– Antibody characteristics
 Quantity
 Specificity
 Thermal amplitude
 Ability to fix compliment
 Ability to bind tissue macrophages
– Antigen characteristics
 Density of antigen on cell surface
 Degree of antigen expression
 Patient age

(AIHA)
Diagnosis
 Serologic evidence of the presence of an antibody:
Coombs Test
– Direct antiglobulin test (Direct Coombs Test)
 Patient’s RBC’s mixed with antiglobulin
–Specific focus on IgG and C3d
 Positive in almost all cases
 Non-specific for a RBC autoantibody

(AIHA)
Diagnosis
 Serologic evidence of the presence of an antibody:
Coombs Test
– Indirect antiglobulin test (Indirect Coombs
Test)
 Positive in approximately 80% of cases
 Autoantibodies likely to be panreactive as
compared to alloantibodies

DIRECT AND INDIRECT COOMBS TESTS

(AIHA)
Treatment
Role of Blood Transfusion
 Limit to life threatening situations or high risk patients
 Best to use most compatible units
 Often difficult to match
 Transfusion may stimulate further formation of
autoantibody

(AIHA)
Treatment
Warm AIHA
 Folic acid supplementation
 Glucocorticoids
 Splenectomy
 Cytotoxic drugs
 Rituximab
 Plasmapheresis
 IVIg

(AIHA)
Treatment
Glucocorticoids
 Initial therapeutic intervention for warm AIHA
– Initial prednisone dose of 1-1.5mg/kg/day
 Response rates
– 20-30% have a lasting remission
– 50% require low dose maintenance
– 10-20% do not respond
 Considered a failure if 15mg or greater of prednisone
is required to maintain a Hct of at least 30%

(AIHA)
Treatment
Glucocorticoids
 Multiple possible dverse effects
– Osteoporosis
– Avascular necrosis
– Increased risk for infection
– Cataracts
– Glucose/lipid abnormalities
– Behavioral changes
– Peptic ulcer disease
– Myopathy

(AIHA)
Treatment
Splenectomy
 Second line treatment
 Has multiple benefits
– Removes site of hemolysis
– Decreases antibody production
 Response rate of 60-75%
– Often will still require steroids
 Requires immunization

(AIHA)
Treatment
Cytotoxic Drugs
 Includes cyclophosphamide, azathioprine and
cyclosporine A
 Results in a 40-60% response rate
 May cause bone marrow suppression

(AIHA)
Treatment
Rituximab
 An anti-CD20 monoclonal antibody
 Results in B cell destruction
– Complement mediated cytotoxicity
– Antibody dependent cytotoxicity
– Inhibition of B cell proliferation
– Induction of apoptosis

(AIHA)
Treatment
Plasmapheresis
 Current data based on case reports
 Has been difficult to assess effectiveness in setting of
other treatments
 May be useful in fulminant cases until other therapies
can take effect

(AIHA)
Treatment
Intravenous immunoglobulin
 Used in AIHA after it was found to work well for
patients with ITP
 Second line therapy for steroid non-responders
 When used, requires higher doses than used for
treatment of ITP

ANEMIA (AIHA)
Treatment
 Avoidance of cold temperature
 Folic acid supplementation
 Cytotoxic drugs
 α-interferon
 Plasmapheresis
 Rituximab
 Efforts should be made to limit transfusion
– Difficult to match
– Transfusion worsens hemolysis
– Blood warmer may reduce the risk of further hemolysis

Q no 1: Patients from which of the following regions need not
be screened for glucose-6-phosphate dehydrogenase (G6PD)
deﬁciency when starting a drug that carries a risk for G6PD
mediated hemolysis?
A. Brazil
B. Russia
C. Southeast Asia
D. Southern Europe
E. Sub-Saharan Africa
F. None of the above

Q no 2: You are asked to review the peripheral blood smear
from a patient with anemia. Serum lactate dehydrogenase is
elevated and there is hemoglobinuria. This patient is likely to
have which physical examination ﬁnding?
A. Goiter
B. Heme-positive stools
C. Mechanical second heart sound
D. Splenomegaly
E. Thickened calvarium

Q no 3: The triad of portal vein thrombosis, hemolysis,
and pancytopenia suggests which of the following
diagnoses?
A. Acute promyelocytic leukemia
B. Hemolytic-uremic syndrome (HUS)
C. Leptospirosis
D. Paroxysmal nocturnal hemoglobinuria (PNH)
E. Thrombotic thrombocytopenia purpura (TTP

Q no 4: All of the following laboratory values are
consistent with an intravascular hemolytic anemia except
A. Increased haptoglobin
B. Increased lactate dehydrogenase (LDH)
C. Increased reticulocyte count
D. increased unconjugated bilirubin
E. increased urine hemosiderin

Q no 5: Which of the following hemolytic anemias can be
classiﬁed as extracorpuscular?
A. Elliptocytosis
B. Paroxysmal nocturnal hemoglobinuria
C. Pyruvate kinase deﬁciency
D. Sickle cell anemia
E. Thrombotic thrombocytopenic purpura

Q no 6 :During the winter months, a 65-year-old man
presents with livedo reticularis and purple fingertips.
Other symptoms include arthralgia and weakness. Renal
impairment is present on laboratory testing. Which of the
following is the most likely diagnosis?
A. Cold agglutinin disease
B. Henoch-schönlein purpura
C. Antiphospholipid antibody syndrome
D. Cryoglobulinemia
E. Cholesterol embolic disease

Q no 7: A 21-year-old woman is suspected of having
mycoplasma pneumonia based on symptoms of a dry
cough, fever, normal lung examination but abnormal
chest x-ray with bilateral infiltrates. She is also anemic
with hemoglobin of 10.5 g/dL, reticulocyte count 7%, and
WBC 12,000/mL. Hemolytic anemia from cold agglutinins
is suspected as the cause. Which of the following tests will
confirm autoimmune causation as the cause of the
anemia?
A. Positive antinuclear antibody (ANA)
B. Positive rheumatoid factor
C. Polyclonal gammopathy
D. Presence of heinz bodies
E. Positive Coombs’ test

Q no 8: A 19-year-old man had his spleen removed a year
ago after a motorcycle accident. Which of the following
findings on the blood film are most consistent with a
previous history of splenectomy?
A. Increase in macrophages
B. Leukopenia
C. Polycythemia
D. Increased reticulocytes
E. Red cells with nuclear fragments

Q no 9: A 57-year-old man, with a history of chronic
alcohol ingestion, is admitted to the hospital with acute
alcoholic intoxication and lobar pneumonia. Physical
examination reveals pallor; a large, tender liver; and
consolidation of the right lower lobe. Laboratory data
include hemoglobin of 7 g/dL, WBC of 4000/mL, and
platelet count of 85,000/mL.Which of the following is the
most likely factor for the anemia?
A. Hemolysis
B. Hemobilia
C. Vitamin B12 deficiency
D. Toxic marrow suppression
E. Hemoglobinopathy

Q no 10 : A 35-year-old female who is recovering from
Mycoplasma pneumonia develops increasing weakness.
Her Hb is 9.0 g/dL and her MCV is 110. Which of the
following is the best test to determine whether the
patient has a hemolytic anemia?
A. Serum bilirubin
B. Reticulocyte count and blood smear
C. Mycoplasma antigen
D. Glucose phosphate dehydrogenase level
E. Liver spleen scan

KEYS
 Q no 1 : B
 Q no 2 : C
 Q no 3 : D
 Q no 4 : A
 Q no 5 : E
 Q no 6 : D
 Q no 7 : E
 Q no 8 : E
 Q no 9 : D
 Q no 10 : B

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